Detection and quantification of human herpes virus 7 by enzymic amplification

ABSTRACT

The present invention relates to an isolated nucleic acid molecule comprising: (i) a primer protion consisting of a contiguous sequence of from 10 to 50 nucleotides capable of hybridizing to (a) the target nucleic acid molecule represented by SEQ ID NO:1, or (b) to the complementary stand thereof; and optional (ii) a further portion comprising from 1 to 25 nucleotides joined to and immediately 5′ to the 5′ end of the primer portion.

This invention was made with US government support under grant numberA133389-02 (“significance of HHV6-7 for immunocompromised patients”)awarded by the National Institutes of Health, Bethesda, Md. 20892, USA.The US Government has certain rights in the invention.

This invention relates to a method for detecting and quantifying thepresence of human herpesvirus 7 (HHV-7) in a sample, and to nucleic acidsequences useful in such a method.

HHV-7 is a member of the family Herpesviridae and is related to butgenetically distinct from human herpesviruses 6A and 6B (HHV-6A andHHV-6B). HHV-7 was first isolated in 1989 and subsequently found to bepresent in the saliva of from 75% to over 90% of healthy adult humans(Frenkel et al (1990): PNAS 87, 748-752; Wyatt et al (1991): J. Virol.65(11), 6250-6265).

HHV-7 has been found to cause an exanthem subitum-type illness in somechildren, one symptom of this illness being the development of rashesand acute febrile illness. At present, other disease symptomsattributable to HHV-7 have not been identified but, by analogy withother human herpesviruses, HHV-7 can be expected to cause disease,especially in immunocompromised hosts, such as organ transplantrecipients. Therefore, it is desirable to be able to detect and quantifythe presence of HHV-7 in samples of human tissue and bodily fluids.Until now, methods of detecting HHV-7 have been serological in nature,relying on the use of antibodies to HHV-7 proteins. These methods are,however, unsatisfactory. This is because they fail to quantify HHV-7, orhave low specificity for HHV-7 with respect to other viruses such asHHV-6A/B.

The inventors have identified a sequence of HHV-7 DNA that is unique tothis virus and not found in other related viral genomes although itcodes for a protein that has some sequence homology with the product ofKA3L gene of HHV6. The unique sequence is a 193 base pair fragment ofHHV-7 DNA, and was identified by digestion of purified HHV-7 DNA with arestriction endonuclease, followed by cloning the restriction fragmentsinto a plasmid vector. Using the sequence of this 193 base pair “target”fragment, it has been possible to synthesise a variety of primers thathybridise to it over part of its length. These can be used in PolymeraseChain Reaction (PCR), including nested PCR reactions, to amplify the 193base pair fragment.

The inventors have also prepared a mutant DNA control sequence based onthe 193 base pair fragment. This construct has internal substitutionsthat provide an additional restriction site (for the restrictionendonuclease SmaI) that is absent from the target sequence. Primerssuitable for construction of the mutant sequence by PCR amplificationhave also been synthesised.

The mutant control sequence can therefore act as a target sequencemimetic for the 193 base pair fragment in any PCR reaction performed ona sample containing the 193 base pair sequence and the control sequence.This forms the basis of a method of detecting and quantifying thepresence of the 193 base pair fragment in samples, for example samplesof human tissue or bodily fluids, and thus quantifying HHV-7 in thesesamples. A known amount of the control sequence is added to the sample;a pair of primers is also added and PCR is performed. As the target andcontrol sequences are very similar, and preferably identical at theprimer binding sites, one pair of primers allows the amplification ofboth the 193 base pair target sequence and the control sequence by PCR.Cleavage of the mutant sequence with the restriction endonuclease SmaI,which does not cleave the 193 base pair wild-type sequence, followed byquantification of the cleavage products then allows the amount of thetarget sequence present to be determined as the amount of the controlsequence added is known. This, in turn, allows the presence of the HHV-7virus in the sample be quantified as each HHV-7 genome comprises onetarget sequence.

As the target sequence is unique to HHV-7, this method can be used toquantify HHV-7 accurately in samples, thus potentially providinginformation as to the likelihood of the sample donor suffering from thesymptoms caused by the virus.

Accordingly, the invention provides:

An isolated nucleic acid molecule comprising (i) a primer portionconsisting of a contiguous sequence of from 10 to 50 nucleotides capableof hybridising to (a) the target nucleic acid molecule represented bySEQ ID NO:1 or (b) to the complementary stand thereof; and optionally(ii) a further portion comprising from 1 to 25 nucleotides joined to andimmediately 5′ to the 5′ end of the primer portion;

a pair of nucleic acid molecules as defined above wherein one nucleicacid molecule has a primer region of type (a) as defined above and theother nucleic acid molecule has a primer region of type (b) as definedabove; and wherein the two primers in combination are capable ofamplifying the target nucleic acid molecule represented by SEQ ID NO:1or a section thereof in a polymerase chain reaction;

a pair of nucleic acid molecules as defined above that hybridiserespectively to the target nucleic acid sequence of SEQ ID NO:1 and itscomplementary stand in such positions that the 5′ end of the each primerregion hybridises at a location that is from 0 to 50 nucleotides 3′ tothe 5′ end of the sequence of SEQ ID NO:1 or the complementary strandthereto;

A method of determining the amount of a target nucleic acid having thesequence shown in SEQ ID NO:1 in a sample which method comprises:

(i) mixing the sample with a predetermined amount of control nucleicacid;

(ii) bringing the mixture formed in (i) into contact with at least onenucleic acid primer as defined above that is capable of hybridising tothe target nucleic acid and at least one primer as defined above that iscapable of hybridising to the control nucleic acid;

(iii) performing a nucleic acid amplification reaction, which reactionrequires the presence of the primers defined in (ii) to amplify thetarget nucleic acid sequence or a section thereof and the controlnucleic acid or a section thereof;

(iv) determining the relative quantities of the amplified control andtarget nucleic acids; and

(v) calculating from the determination of (iv) the amount of targetnucleic acid in the sample;

An isolated nucleic acid sequence selected from the sequences shown inSEQ ID NO:1 and SEQ ID NO:2, or the complementary strands thereof; and

A kit for quantification of human herpesvirus 7 (HV-7) in a sample bymeans of a method as defined above which kit comprises:

(i) a control nucleic acid as defined above;

(ii) one or more pairs of primers as defined above that are suitable foramplifying both the target and control nucleic acid sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Construction of the 183bp control sequence of SEQ ID NO:2.

FIG. 2. Results of qualitative PCR on saliva samples, using the innerand outer primers of the invention.

FIG. 3. Demonstration that the assay of the invention gives accuratedata on samples with known genomic equivalents.

FIG. 4. Prevalence of HHV-7 DNA in blood samples from HHV-7 seropositiveindividuals varies according to the quantity of input DNA used foranalysis.

FIGS. 5 & 6. Longitudinal analysis of HHV-7 viral load in saliva andperipheral blood of healthy volunteers shows that the amount of virusremains relatively constant over time.

FIGS. 7, 8 & 9. Qualitative detection of HHV-7 in live, renal and bonemarrow transplant recipients.

FIG. 10. HHV-6 and HHV-7 blood viral load versus clinical syndrome.

FIG. 11. HHV-7 load in organs at death.

The target sequence that is amplified in methods of the invention is the193 base pair sequence of SEQ ID NO:1 or a section of it, preferably theentire 193 base pair sequence or the section thereof shown in SEQ IDNO:1. Where the target sequence is a section of the sequence of SEQ IDNO:1, the section may be of any length provided that the section inquestion is unique to the HHV-7 genome, in the sense that it is notfound in the genomes of other viruses, particularly HHV-6A and HHV6B,that infect human cells, or in the human genome. For example, a sectionmay comprise up to 50, up to 100, up to 150 or up to 190 of thenucleotides of SEQ ID NO:1.

The control sequence that is amplified in methods of the invention maybe any suitable sequence that allows quantification of the targetsequence. The control nucleic acid may be any suitable nucleic acid, eg.a single or double stranded fragment or a sequence contained within avector, for example a plasmid vector. Preferably, the control nucleicacid is chosen so that it will undergo amplification at a substantiallyidentical rate to that of the nucleic acid in the sample which is to beanalyzed. Following amplification, measurement of the amount ofamplified control sequence relative to the amount of amplified targetsequence from the sample allows the quantity of nucleic acid originallypresent in the sample to be determined.

Accordingly, it is preferred that the control sequence is of similarlength and topology to the target sequence. Thus, the control sequencemay be of any length. For example, lengths of up to 50, up to 100, up to250, up to 200 or up to 300 base pairs are preferred with lengths in theregion of from 100 to 250 bp being more preferred and lengths of 180 to220 bp being particularly preferred. The most preferred controlsequence, as shown in SEQ ID NO:2, has a length of 183 base pairs, as itwas obtained by introducing a SmaI recognition site into the sequence ofSEQ ID NO:1.

Also, it is preferred that a control sequence of the invention has ahigh degree of sequence homology to the target sequence. Preferably, thecontrol sequence has at least 70%, at least 80%, at least 90%, at least95% or at least 99% homology with the target sequence.

Further, it is preferred that a control sequence of the invention has asimilar topology to the target sequence. Thus, linear control sequencesare preferred.

The difference between the target and control sequences may be in thenature of one or more nucleic acid deletions, substitutions orinsertions, or may arise from any other type of nucleic acidrearrangement or sequence alteration.

In a preferred embodiment of the invention, the control nucleic aciddiffers from the target nucleic acid by the presence of at least onepredetermined restriction enzyme recognition sequence (restrictionsite). Such a restriction site can be generated, for example, using acopy of the original target sequence which is altered by site-directedmutagenesis or by using oligonucleotide primers complementary to thetarget sequence at all residues except those which need changing toconstruct the desired restriction site. Such primers may have anyappropriate sequence and the primer pair represented by SEQ ID NO:7 andSEQ ID NO:8 are examples of suitable sequences. These primers can beused to introduce a SmaI site into the sequence of SEQ ID NO:1, andother primers could be used to introduce other restriction sites.Following PCR amplification of the target and control sequences, thereaction mixture (or a portion thereof) is brought into contact with arestriction enzyme capable of cutting the predetermined restriction siteor sites. In this way, the uncut amplified target sequence can beresolved, eg. by electrophoresis, from the two or more fragments of thecut amplified control sequence; ultimately allowing quantification ofthe target sequence.

It is preferred that the predetermined restriction site in the controlnucleic acid is a restriction site that is unique to the controlsequence, i.e. only two fragments of DNA will be produced upon digestionof the control fragment. Desirably, the predetermined restriction siteis centrally located in order that the two fragments produced bydigestion are of the same or of a similar size, eg. within 15,preferably 5, nucleotides in length of each other. This will mean thatthe two fragments produced will appear as a single band on a gelfollowing electrophoresis. Quantitative analysis of the band willprovide a determination of the total amount of amplified control DNA.

Other configurations of restriction sites are also possible. Forexample, the control nucleic acid may contain, for example, 2, 3, 4 ormore restriction sites, one or more of which can be cut following PCR.Such multiple restriction sites may be recognition sites for the sameenzyme or for two or more different enzymes. They may be located in amanner such that digestion with the appropriate enzymes producesfragments or a substantially similar size or of different sizes. Suchconfigurations could be used to provide an internal control for theefficiency of the restriction digest or to provide fragments ofconvenient size for resolution. For example, a restriction site uniqueto the selected region of the target nucleic acid which, when cut, willdivide the selected region into two unequal fragments could beeliminated from part of the control sequence region and the samerestriction site introduced at a central location in the said controlsequence. Following PCR and digestion with an enzyme capable of cuttingthis centrally located site in the control region followed by resolutionby electrophoresis, three different size fragments will be seen. Theamount of DNA in all three fragments may be measured and the amount oftarget DNA quantified. If for any reason the restriction digest isincomplete, a further amplified fragment will be seen. However, thiswill not affect the quantification of target nucleic acid, which can bedetermined simply by the ratio of digested fragments.

A further method of controlling the digest is to add to the sample,prior to digesting, a further DNA fragment containing a restriction sitefor the restriction enzyme which is to be used for digesting of thetarget DNA which is of a size such that it will not, either as acomplete fragment or following digestion, be superimposed over digestedor undigested control or target nucleic acid in the resolution step ofthe method of the invention. This can be used to monitor the efficiencyof the digest. The ratio of undigested to digested further DNA can beused to correct for any incomplete restriction digests.

Although the embodiment of the invention discussed above envisages thatthe control region will be altered to introduce a predeterminedrestriction site, the invention also includes altering the control DNAto eliminate a restriction site, should a convenient restriction siteoccur in the target nucleic acid. Where a suitable restriction siteoccurs in the natural target nucleic acid, primers substantiallyequidistant from this site can be selected in order that thepredetermined restriction site is centrally located in the selectedregion.

In control sequences of the invention, any type of restriction site maybe exploited in the manner outlined above. In one preferred controlsequence, as shown in SEQ ID NO:2, a SmaI site has been introduced. Theprimer portions of these nucleic acids are capable of hybridizing tosections of both the target and control sequences, owing to the highsequence homology between the target and control sequences. Inprinciple, such hybridization may occur with any section of the targetsequence as long as the primer portion is capable of hybridizing to boththe target and control sequences and initiating amplification of them.Preferably, the section over which hybridization occurs comprises atleast 10, more preferably at least 15 to 20, for example 16, 17, 18 or19; at least 30; or at least 50 to 100 nucleotides. More preferably, theprimer portions of the nucleic acids of the invention comprise from 10to 15 nucleotides. The sections of the target sequence with whichhybridization occurs may be located in any part of the target sequence;i.e. they may be within the sequence or at one end of it.

Optionally, the nucleic acids of the invention may also comprise afurther portion, especially if the section to which the primer portionhybridizes is at the 5′ end of the target sequence. The optional furtherportion may therefore comprise nucleotides that extend beyond the end ofthe target or control sequence. The length of the further portion may beup to 25, for example, up to 20, up to 18, up to 10 or up to 5nucleotides. The sequence of the further portion may be of any nature.For example, it may be native HHV-7 nucleic acid sequence or a sequencewith substantial sequence homology thereto. Alternatively, the sequenceof the further portion may be an unrelated (non-HHV-7) sequence. Forexample, the further portion may be an amplifier strand as discussed inEP-A-0-317,077. This will allow detection of HHV-7 DNA by addition of amultimer probe comprising a first nucleic acid sequence complementary tothe amplifier strand and two or more (eg. 4, 5, 6 or 7) sequencescomplementary to a separate labelled nucleic acid probe.

It is preferred that the primer portions will be perfectly complementaryto the section of the target sequence to which they hybridise, in thesense that every nucleotide will base pair with the one with which itpairs most stably (A with T or U; C with G). However, small deviationsfrom this rule may be allowed, so long as they do not prevent the primerfrom hybridising with the target and control sequences and initiatingamplification. Thus, the primer portion may be, for example, up to 70%,up to 80%, up to 90%, up to 95% or up to 99% complementary to therelevant section of the target or control sequence.

Preferred primers capable of hybridising to both the target (SEQ IDNO:1) and control sequences (e.g. SEQ ID NO:2) of the invention are theprimers of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

Preferably, the primers of the invention will be in isolated form, forexample in aqueous solution. Thus, in samples of the primers, at least90% at least 95% or at least 99% of the nucleic acid in the sample willtypically be a single nucleic acid of the invention, or a pair ofprimers capable of amplifying of the target and control sequences in aPCR.

In a preferred embodiment of the invention, pairs of nucleic acids asdescribed above are employed. Any two nucleic acids of the invention mayform such a pair as long as the primer portion of one of them is capableof hybridizing to the target nucleic acid of SEQ ID NO:1 and the primerportion of the other is capable of hybridizing to the complementarystrand to the target sequence, and as long as the two nucleic acids incombination are capable of initiating amplification of the target andcontrol sequences or sections thereof. Thus, in preferred primer pairsof the invention, the two primer portions hybridize with the targetnucleic acid and its complementary strand towards the 5′ end of eachstrand. Accordingly, it is preferred that the nucleic acid moleculeshybridize at a location that is from 0 to 50 nucleotides 3′ to the 5′end of the sequence of SEQ ID NO:1 of the complementary strand thereto.For example, it is preferred that the 5′ end of each primer portionhybridizes at a location that is from 0 to 10, from 10 to 20, from 20 to30, from 30 to 40 or from 40 to 50 nucleotides 3′ to the 5′ end of thesequence of SEQ ID NO:1 or of the complementary strand thereto.

One preferred primer pair of the invention is provided by thecombination of the nucleic acid molecules of SEQ ID NO:3 and SEQ ID.NO:4. Another preferred pair is represented by SEQ ID NO:5 and SEQ IDNO:6.

The invention further provides methods for detecting and quantifying theoccurrence of HHV-7 in samples. Typically, these samples will comprisehuman tissue, cells or bodily fluids or extracts derived from suchtissues, cells or fluids. Thus, for example, methods of the inventionmay be used to detect and/or quantitate the number of viral genomespresent in a sample, quantitative methods being preferred.

Thus, methods of the invention can detect and or quantify HHV-7 under avariety of circumstances. For example, it is desirable to detect and/orquantify HHV-7 in samples taken from patients who may be infected withHHV-7. Also, methods of the invention can be used to evaluate theeffectiveness of antiviral drugs. In this case, cell cultures comprisingHHV-7 can be assayed using methods of the invention, the resultinginformation being used to determine what concentration of antiviralagent to use.

Methods according to the invention rely on amplifying target and controlnucleic acids as described above by any suitable method, PCR being apreferred method. In such methods, one or more primers as describedabove are used to initiate amplification. Typically, primers areemployed in pairs for the amplification of double stranded nucleic acidwith one primer binding to a site towards the 5′ end of each strand.

In general, primers, preferably primer pairs as described above, areadded to the sample, as well as the control sequence. As the target andcontrol sequences have high sequence homology, and are preferablyidentical at the sites to which the primers bind, one pair of theprimers hybridises to both target and control sequences. Thus, PCR leadsto amplification of both sequences.

For the analysis and quantification of the target nucleic acid sequenceor section thereof, the predetermined amount of control sequence addedmay be any suitable amount, preferably an amount within a factor of10³-fold of the amount of target nucleic acid suspected to be containedwithin the sample. In a typical reaction it is likely that between 5 and10⁵ molecules of control sequence, eg. about 1000, will be used,although those of skill in the art will appreciate that this may vary,depending, for example, on the concentration of nucleic acid in thesample being analyzed. In order to achieve a more accurate result, aseries of reactions each using a different amount of control sequencemay be performed and the results compared.

Amplification, preferably by means of PCR, is then carried out. WherePCR is employed, any number of amplification cycles that amplifies thetarget sequence to a sufficient degree may be used, for example up to20, up to 40 or up to 80 cycles, with from 30 to 50 cycles beingpreferred and 35 to 45 cycles being particularly preferred in the firstround of PCR. Similarly, any suitable conditions may be used,particularly as regards types and concentrations of reagents used in thePCR reactions and temperatures in the denaturation, annealing andextension stages of the PCR reaction.

In a particularly preferred embodiment of the invention, a nested PCRreaction is used to amplify the target and control sequences. In nestedPCR, two or more pairs of primers are employed sequentially to amplifyprogressively shorter sections of the target and control sequences.Thus, in a two-stage nested PCR, two pairs of primers are used. Thefirst pair hybridises to the target or control nucleic acid towards therespective 5′ ends of each strand. Amplification results in a nucleicacid product that is equivalent the section of the target or controlsequence between the 5′ ends of the two primers. A further pair ofprimers is then employed. These bind to the amplified product at sitesthat are 3′ to the first primer binding sites and are thus within thefirst pair of primer binding sites. Thus, the nucleic acid molecules ofthe primer pair used in each successive amplification routine arecapable of hybridizing to the target sequence of SEQ ID NO:1 in such aposition that the 5′ ends of the primer regions are located at least onenucleotide 3′ to the site at which the 5′ end of the correspondingprimer of the previous pair hybridized. Preferably, the 5′ end of eachsuccessive corresponding primer should hybridize to the target sequenceat least 5, at least 10, at least 20 or at least 30 nucleotides 3′ tothe site at which the 5′ end of the previous corresponding primerhybridized.

A second or subsequent amplification stage results in a shorter nucleicacid section than was obtained in the first amplification. The productsof the second stage are then detected and quantified.

Nested PCR has the advantage that a greater total number of cycles ofamplification can be performed without significant amplification ofbackground contaminants (which are amplified to a small extent in manyPCR reactions owing to non-specific binding of primers to sequencesother than the one of interest).

This background amplification places a practical limit on the number ofamplification cycles that can be achieved with any single primer pair.Nested PCR enables this limit to be overcome and lower levels of targetsequence to be detected. This is particularly beneficial in clinicalapplications where HHV-7 may be present at very low levels in humantissues or bodily fluids. As an example of primers suitable for nestedPCR according to the invention, primers HHV7-1 (SEQ ID NO:3) and HHV7-2(SEQ ID NO:4) are suitable as the first (outer) pair of primers as theyhybridise to sites close to the 5′ ends of the two strands of the targetsequence (SEQ ID NO:1). Primers HHV7-3 (SEQ ID NO:5) and HHV7-4 (SEQ IDNO:6) are suitable as the second (inner) pair of primers as theyhybridise to sites further from the 5′ ends of the two strands of thetarget sequence and more towards the centre of the target sequence.Second round amplification with these primers may occur for 5 to 40cycles, with from 5 to 25 cycles being preferred and 12-20 cycles beingparticularly preferred.

Analysis of the products of the amplification reaction is then carriedout. Quantitative analyses are preferred, though detection of the targetnucleic acid sequence without quantification is also within the scope ofthe invention. Detection can, for example, be achieved using a singleprimer sequence of the invention as a probe. A single primer sequencewill not act to amplify the target sequence but will, if it issufficiently complementary, hybridise specifically to it. Thisspecificity can be exploited, by any means known in the art (such asthose of Sambrook et al; 1989, Molecular Cloning: A Laboratory Manual),to detect the presence of the target nucleic acid. For example, theprobe may carry a label (e.g. a radioactive or fluorescent label) whichcan be detected by a suitable method.

Detection and quantification of the amplified target and control nucleicacids may be performed by any suitable means known in the art. However,certain methods of detection and quantification are preferred. Inparticular, methods relying on the presence of a unique restriction sitein either the target or control sequence are preferred, as describedabove. In such methods, one sequence, typically the control sequence,includes one or more restriction sites that are absent from the othersequence. Following amplification, one or more of these distinctiverestriction sites is cleaved with the appropriate restriction enzyme.The cleavage products are then identified and quantified, thus allowingquantification of the target sequence. Alternatively, the presence ofthe altered base can be detected by a point mutation assay (Kaye et al(1992) J. Virol. Methods) to yield the distribution of bases at thespecific position represented by target or control sequence amplicons.

Preferably, the PCR amplified target and control nucleic acids orfragments thereof, such as cleaved fragments of the control nucleicacid) are resolved by electrophoresis through other methods, eg. othertypes of size-fractionation may be used if appropriate.

Following resolution of the target and control nucleic acids, therelative amounts of these nucleic acids may be measured by any meansknown in the art. Preferably, the amplified nucleic acids will contain aradioactive label or fluorescent label which can be measured by, forexample, autoradiography followed by scanning densitometry or laserdetection of the amplified products. The radio label may be present inthe primers, or incorporated in the nucleotides used in nucleic acidsynthesis during amplification reaction.

Alternatively, the amplified target and control nucleic acids can beresolved by use of oligonucleotide probes which are specific for one orthe other, but not both, amplified products. As the control nucleic acidtypically differs slightly from the target sequence, it is possible tosynthesise probes that are specific for the amplified control nucleicacid but do not, under conditions of high stringency, hybridise to thetarget nucleic acid. A second probe specific for the target sequencecould be used to quantify that sequence. Alternatively, a first probespecific for the target or control nucleic acid could be used todetermine the quantity of one or other of these products and then asecond probe specific to both products used on the same sample to ensurethe total amount of nucleic acid produced. The difference between themeasurement with the first probe and measurement with the probe commonto both products will provide an indication of the amount of whicheverof the selected and control products was not measured in the firstmeasurement. In a further embodiment, where the difference between thecontrol and target nucleic acids is small, a single oligonucleotideprobe encompassing the region of difference and 100% homologous to asection of one or other of the two sequences could be used. At lowstringency, the probe will hybridise to both the control and selectedregions although at high stringency it will only hybridise to a regionof 100% homology.

When amplification by PCR is used, the control and target nucleic acidswill be substantially homologous. For example this may mean that besidesthe differences mentioned above which allow resolution of the amplifiedselected and control nucleic acids, there will be no other differencesbetween the sequences. This will help to ensure equal efficiency of thereaction on both control and target sequences. However, minoralterations to the sequences may be possible without detriment to thepresent invention.

The nucleic acids which may be analyzed include DNA and RNA, with DNAbeing preferred. When RNA is being amplified by PCR, an initial reactionusing a first primer and reverse transcriptase is required. Theefficiency of reverse transcription can vary very significantlydepending upon reaction conditions. Therefore, when the target nucleicacid is RNA, it is preferred that the control nucleic acid be RNA. Suchcontrol RNA can be generated using a control DNA sequence cloned into anRNA transcription vector which generates an RNA species using a suitableRNA polymerase in conjunction with a suitable promoter, eg a T3 or T7promoter. The control RNA generated can be quantified using standardspectrophotometric assays. The advantage of such a system is that theefficiency of both the reverse transcription and PCR steps arecontrolled for.

The invention further provides isolated nucleic acid sequences relatedto the target and control sequences. The target and control sequencesthemselves are given in SEQ ID NO:1 and SEQ ID NO:2 . The section of SEQID NO:1 amplified by the primers of SEQ ID NO:3 and SEQ ID NO:4 is givenin SEQ ID NO:9, and the 143 bp section of SEQ ID NO:1 amplified byprimers of SEQ ID NO:5 and SEQ ID NO:6 is given in SEQ ID NO:10. Themost preferred control sequence of the invention is given in SEQ IDNO:2. The section of SEQ ID NO:2 amplified by the primers of SEQ ID NO:5and SEQ ID NO:6 is given in SEQ ID NO:11.

The invention also provides kits for quantification of HHV-7 in samplesas described above. These kits comprise reagents suitable for performingmethods as described above, and therefore for effecting detection, andpreferably quantification, of HHV-7 in samples. A kit suitable forquantifying HHV-7 in a sample will. comprise a control nucleic acid, forexample the control nucleic acid of SEQ ID NO:2, as described above; andone or more pairs of primers as described above that are suitable foramplifying both the target and control nucleic acid sequences, orsections thereof. Preferred kits according to the invention comprise acontrol nucleic acid having a restriction site not present in the targetnucleic acid, as described above; for example the control nucleic acidof SEQ. ID. NO:2, which contains a SmaI site.

Preferred kits of the invention may also comprise one or more preferredprimer pairs such as the primer pair represented by SEQ ID NO:3 and SEQID NO:4 and/or the primer pair represented by SEQ ID NO:5 and SEQ IDNO:6. Particularly preferred kits of the invention will be suitable forperforming nested PCR methods employing two or more pairs of primers.Thus, for example, a preferred kit according to the invention maycomprise the primer pair represented by SEQ ID NO:3 and SEQ IDNO:4together with the primer pair represented by SEQ ID NO:5 and SEQ IDNO:6. Typically, these will be provided in separate containers as theywill be used in separate stages of a method according to the invention.

Kits according to the invention may also comprise any other suitablereagents, for example a restriction enzyme, such as SmaI, suitable forcleaving a control sequence comprising a restriction site not found inthe target nucleic acid.

Nucleic acids of the invention are preferably DNA although other nucleicacids such as RNA may be used.

The 193 base pair fragment of the invention encodes a polypeptidehomologous with the KA3L gene of HHV-6. By extension, it is thereforethe positional homologue of the Herpes Simplex Virus ICP27 gene. HSVICP27 has been shown to be an important gene in the regulation of mRNAtranscripts and it is therefore a potential antiviral target, since HSVICP27 deletion mutants do not propagate in cell culture.

Therefore, the 193 bp sequence, and the sequences of the invention thatare related to it, may be useful in interrupting viral transcription.Accordingly, nucleic acid fragments having the sequences of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and thecomplementary strands thereof, may form the basis of antiviral agents.

In particular, they may be used to prepare “antisense” DNA constructswhich, when transcribed as mRNA, hybridise to the “sense” mRNAtranscribed from the corresponding native viral gene, typically a HHV-7gene, thus forming a duplex that cannot be translated into a protein andis degraded by the host cell. Thus, the expression of the native viralprotein is reduced, regulation of mRNA transcripts is disrupted, andviral replication in the host cell is impaired.

For in vivo activity, antisense oligonucleotides will requiremodifications in the molecule's backbone and possibly additions ateither the 5′ or 3′, or both, ends of the molecule. This is to (1)achieve resistance to degradation by DNAases, (2) enhance the potency ofthe molecule and (3) to enhance uptake of the oligonucleotide by cells.This is an area of intense research and development. Currently thetechnology exists to produce sufficient quantities of modifiedoligonucleotides for therapeutic use (see for example Biotechnology,November 1993, page 1225). A number of different types of modificationto oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones (where the phospategroups in the nucleic acid backbone are replaced by methylene orsulphur) and addition of acridine or polylysine chains at the 3′ and/or5′ end of the molecule. For the purpose of the present invention, it isto be understood that all or part of the oligonucleotides describedherein may be modified by any method available in the art in order toenhance their in vivo activity or lifespan.

Further, it will be clear to those of skill in the art that specificoligonucleotides described herein are represented in standard notation,ie., by the letters A, C, G or T (or U) to indicate the base of thenucleotide, written in the 5′ to 3′ direction. Unless specified to thecontrary, these sequences may be modified as described above.

The nucleic acid fragments (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10 and SEQ ID NO:11, of the invention may be used as thebasis for antiviral agents against any virus that has a suitablesequence in its genome, preferably a human herpesvirus, more preferablyHHV-7.

The invention therefore provides: nucleic acid fragments of theinvention for use in methods of treatment of the human or animal body bytherapy, particularly with a view to treating, or preventing orameliorating pathologies caused by viruses, especially humanherpesviruses, such as HHV-7; use of nucleic acid fragments of theinvention in the manufacture of medicaments for the treatment,prevention or amelioration of such viral pathologies; methods oftreating, preventing or ameliorating these viral diseases byadministering nucleic acid sequences of the invention alone or incombination with a pharmaceutically acceptable carrier, diluent orexcipient to human or animals in need thereof; and pharmaceuticalcompositions comprising nucleic acid sequences of the invention and apharmaceutically acceptable carrier, diluent or excipient.

The following examples illustrate the invention.

EXAMPLES Example 1 Construction of Viral DNA Library

Viral DNA was extracted from infected cells by the method of Mukai et al(1994) J. Med. Virol. 42, 224-227). Virus-infected cells were lysed andtotal nucleic acid digested with RNase a and DNase 1.Trichlorotrifluoroethane was added and mixed vigorously, after which theprecipitate was pelleted by centrifugation at 400 g for 5 min.Supernatant containing viral capsids was layered onto a discontinuous5%/40% glycerol gradient and centrifuged at 100,000 g for 90 minutes.The pellet was digested for 3 hours at 65° C. using proteinase K in a 10mM Tris.HCl, lmM EDTA, 0.1% SDS buffer; yielding viral DNA which waspurified by standard phenol:chloroform extraction and ethanolprecipitation (Sambrook et al (1989) Molecular Cloning; a laboratorymanual (2nd Ed): Cold Spring Harbour Laboratory Press, New York). Threemicrogrammes of viral DNA was digested with 20 units HindIII at 37° C.for 18 hours in a volume of 50 μl Ligation of fragments in pUC18 wasachieved using a commercially-available kit (Ready-To-go, Pharmacia,England) according to the manufacturer's protocol. Competent bacterialhost E. coli was transformed by standard protocols (Sambrook et al), andquantities of cloned HHV-7 DNA were used for sequence determination.Sequencing was performed by the dideoxy method of Sanger using M13universal primers in standardized protocols (Sambrook et al. Comparisonof sequences with those of other herpesviruses was carried out using theGenetics Computer Group software examination of GENBANK gene databases.

Example 2 (i) PCR Primers and Reaction Conditions

A suitable clone, 193 bp in length and named 1D3 (SEQ ID NO:1) wasselected and primer sequences delineating 183 bp (outer primers: SEQ IDNO:3 and SEQ ID NO:4) and 143 bp (inner primers: SEQ ID NO:5 and SEQ IDNO:6) of this sequence were synthesized (R&D Systems, Abingdon,England). PCR buffer composition was 16 mM (NM₄)₂50₄, 67mM Tris-HCl (pH8.8 at 250° C., 0.010% Tween-20 (Bioline Ltd, London/England); 2 mMMgSO₄; 100 ng each primer; 200 μM dATP, dCTP, dGTP, dTTP; and 1 unit Taqpolymerase (“BioTaq”, Bioline, London, England). Total reaction volumewas 50 μl with 90 μl mineral oil overlay. Two rounds of PCR wereperformed, the first using the outer primers and the second using theinner primers. Thermal cycling conditions were the same for both roundsof PCR: 1 cycle at 95° C. for 6 min; 39 cycles at 94° C., 50° C., 72° C.for 30 seconds each followed by 72° C. for 10 minutes.

(ii) Construction of Control Sequence

Mutational primers HHV7-MUT5 (SEQ ID NO:7) and HHV7-MUTS (SEQ ID NO: 8)were designed spanning a suitable internal area (SEQ ID NO:2) of, butnot entirely complementary to, the 1D3 sequence. Separate PCR reactions,employing respective outer primers and inner mutational primers (forexample HHV7-1 and HHV7-MUT6) were optimized and used to generateamplimers with overlapping sequences of the original 1D3 clone but witha SmaI site introduced FIG. 1, A). The two reaction products were mixedin equimolar proportions (FIG. 1, B), denatured at 95° C. for 10 minutesand allowed to reanneal at room temperature for 30 minutes FIG. 1, C).Klenow fragment of DNA polymerase was used to synthesize thecomplementary strand from the overlap in the presence of 200 μM dNTPsand enzyme buffer in a total volume of 100 μl (FIG. 1, D). Afterseparation of the complete double-stranded product from excess primers,enzyme and dNTPs by using a commercially-available column (Qiaquick,Qiagen, England), the construct was further amplified by using the twocomplementary outer primers FIG. 1, E). After similar purification theconstruct was ligated into the pUC18 plasmid vector (Ready-to-go,Pharmacia, England) followed by transformation of competent E. coli bystandard protocols (Sambrook et a. Characterisation of suitable clonesconsisted of size analysis of excised fragments, linearization ofplasmid by Sma1 digestion, and sequencing of the inserts by the dideoxymethod of Sanger (Sambrook et al.

(iii) Qualitative PCR on Saliva Samples (see Table 1)

PCR was performed, using the conditions described in (i) above, onsaliva samples from 5 patients. The results are shown in FIG. 2.Following one round of amplification with the outer primers of SEQ IDNO:3 and SEQ ID NO:4, a band was identified on an agarose gel. Thiscorresponded in size to the 183 bp fragment expected to result from theamplification.

Following a second round of amplification with the inner primers of SEQID NO:5 and SEQ ID NO:6, a band corresponding in size to the expected143 bp fragment was strongly visualized on an agarose gel in all but oneof the samples. This shows that the primers and methods of the inventionreadily detect HHV-7 DNA in practice.

All patients except the PCR negative individual were positive for HHV-7antibodies using an immunofluorescence assay based on patient serareactivity against HHV-7 injected SupTl cells. Primers were used atstandard protocol amounts (100 ng) and 30 μl of whole saliva was used ina 50 μl total PCR mixture.

TABLE 1 DETECTION OF HHV-6 AND HHV-7 IN SALIVA FROM HEALTHY VOLUNTEERS:COMPARISON WITH ANTIBODY STATUS HHV-6 HHV-7 Volunteer SALIVA SERO-SALIVA SERO- No. PCR STATUS PCR STATUS 1 + + + + 2 + + − − 3 + + + +4 + + + + 5 + + + + 6 − − + + 7 + + + + 8 + + + + 9 + + + + 10 + + + +11 + + + + 12 + + + + 13 + + + + 14 + + + + 15 + + + + 16 + + + +

Thirty microliters saliva tested by nested PCR for HHV-6 for HHV-6 andHHV-7. Sera screened at a dilution of 1:10 by indirect immunoflurescenceassays.

Example 3 Standard curve and reproducibility of the quantitative assay.

The ability of the HHV-7 quantitative assay to give accurate data onsamples with known genomic equivalents is illustrated in FIG. 3. In thisexperiment, known copy numbers of plasmid containing the wild-typetarget ranging from 10 to 1,000,000 copies were co-amplified with copynumbers of control sequence in the appropriate range (10-fold more orless than the target sequence). The calculated wild-type copy number wasthen plotted against the actual input copy number, with each data pointrepresenting a mean of three experiments. Linear regression analysisshowed that the calculated and actual copy numbers were highlycorrelated (R=0.994; p<0.001).

Example 4 Cross-sectional and Longitudinal Analysis of HHV-7 ViralBurden in Blood

The prevalence of HHV-7 DNA in blood samples from HHV-7 seropositiveindividuals varies according to the quantity of input DNA used foranalysis. Therefore, at inputs of 10 to 15 ng PBMC DNA the prevalence ofHHV-7 DNA is between 10% and 12% of the seropositive population, whereaswhen 1 μg of input DNA is used, 90% of seropositive individuals werefound to contain HHV-7 DNA in their blood. These data are summarised inFIG. 4. Using the PCR conditions inherent within this patentapplication, these results have implications for the detection of HHV-7DNA in normal and in immunocompromised hosts to detect levels of viralDNA commensurate with reactivation/reinfection, rather than detection oflatent viral DNA.

Longitudinal analysis of HHV-7 viral load in the saliva and peripheralblood of healthy volunteers showed that the amount of virus remainsrelatively constant over time (see FIGS. 5 and 6).

Example 5 Detection of HHV-7 DNA in the Blood of ImmunocompromisedPatients

The qualitative and quantitative PCR methods described in this patenthave been used to detect and measure HHV-7 in liver, bone marrow andrenal transplant recipients, and HIV-infected patients.

a) Qualitative presence of HHV-7 DNA in 40 ng of peripheral bloodsurveillance samples from immunocompromised patients.

i) HHV-7 in transplant recipients

The results of the qualitative detection of HHV-7 in liver, renal andbone marrow transplant recipients on a sample and patient basis areshown in FIGS. 7, 8 & 9, respectively. For comparative purposes, thedetection rates for HHV-5 (cytomegalovirus) and HHV-6 are also shown inthese Venn diagranms. HHV-7 was detected in 26/50 (52%) liver patients,29/60 (48%) renal patients and 16/50 (34%) of the bone marrow transplantrecipients. On a sample basis, 44/473 (9.3%) of samples from the liverpatients, 53/729 (7.4%) of samples from the renal transplant patients,and 22/651 (3.4%) of samples from bone marrow transplant recipients werepositive for HHV-7.

ii) HHv-7 in HIV-infected individuals.

The prevalence of HHV-7 in patients with HIV infections stratifiedaccording to CD4 cell numbers is shown in Table 2. A reduction in theoverall prevalence of HHV-7 DNA in the peripheral blood was observed onprogressing from a CD4 strata of >700 μL to <200 μL.

TABLE 2 DETECTION OF HHV-6 OR HHV-7 IN 473 SAMPLES COLLECTED FROM 160HIV-POSITIVE PATIENTS Number (%) samples PCR- CD4 cells per cubicmilimeter positive for: <200 200-400 400-700 >700 TOTALS HHV-6 8*/1363/164 9/129 2/44 22/473 (5.9%)  (1.8%) (6.9%) (4.5%) (4.6%) HHV-7 3*/1361/164 4/129 3/44 11/473 (2.2%) (0.61%) (3.1%) (6.8%) (2.3%)

*One sample PCR-positive for both HHV-6 and HHV-7

b) HHV-7 load in liver transplant recipients

The maximum virus load detected during surveillance of liver transplantrecipients is shown in FIG. 9. Maximum viral loads for HHV-7 weresimilar to those for HHV-6, both of which were substantially lower thanfor CMV.

c) Clinicopathological Correlations of HHV-7 infection in livertransplant recipients

In contrast to other betaherpesviruses, our investigations have shownvery few pathological consequences of HHV-7 infection in the livertransplant recipients to date.

Example 6 Detection and Quantification of HHV-7 DNA in Children withFebrile Illness

Forty children aged between 6 months - 24 months with febrile illnesshave been investigated for the presence of HHV-7 DNA as a marker ofprimary infection relating to the clinical appearance of exanthemsubitum-like disease. Six of these children had HHV-7 DNA in theirblood, with a median viral load of 4,300,000 genomes/ml (range57,000-290,000,000; FIG. 10). Careful inspection of the results of PCRon saliva from these individuals revealed that 3 were highly likely tobe experiencing primary infection with HHV-7. These 3 children possessedthe highest viral load of the group (median 32,000,000 genomes/ml),which was approximately 100-fold higher than the median viral load forHHV-6 in children undergoing primary HHV-6 infection (138,000 genomes/mlof blood).

Example 7 Prevalence and Quantity of HHV-7 in Necropsy Tissue from AIDSPatients and Control Subjects

DNA extracts (10 μg) from organs obtained at necropsy were used as asubstrate for HHV-7 DNA analysis by the qualitative and quantitative PCRmethods described in this patent. Although HHV-7 DNA was detected in59/125 (47%) of tissues from AIDS patients, it was at a significantlylower viral load than that presenting in control subjects (median HHV-7load in controls 24 genomes/μg DNA; range 0 to 550,000), whereas in AIDSpatients (median =0 genomes/μg DNA; range 0 to 295; Mann-Whitney test:p<0.001). These data are shown in FIG. 11.

Sequence Information

Target and Control Sequences

SEQ ID NO:1. 193 bp Target Sequence

AAGCTTTTTA CATTTGGCTT GCTTTTTGGT TTGTAAATTC AATTGGACGG TTTGCTTAGATTGCTGTGAA GCAAAGCTGC AAGACGGAGT TGTAGAACAT GCAACATTAA AGCTGTTGTACTCTTCAGGC ATGTTAGGAT GCAGACCAAA CTCCATAAAT TCTTTGGGAA GATAGGTACAGAAATATAAG CTT

SEQ ID NO:2. 183 bp Control Sequence

TTTTTACATT TGGCTTGCTT TTTGGTTTGT AAATTCAATT GGACGGTTTG CTTAGATTGCTGTGAAGCAA AGCTGCAACC CGGGGTTGTA GAACATGCAA CATTAAAGCT GTTGTACTCTTCAGGCATGT TAGGATGCAG ACCAAACTCC ATAAATTCTT TGGGAAGATA GGTACAGAAA TAT

Outer (first round) Primers

HHV7-1 5′ TTT TTA CAT TTG GCT TGC TTT TTG 3′ (SEQ ID NO:3)

HHV7-2 5′ ATA TTT CTG TAC CTA TCT TCC CAA 3′ (SEQ ID NO:4).

Inner (second round) Primers

HHV7-3 5′ TGC TTT TTG GTT TGT AAA TTC 3′ (SEQ ID NO:5)

HHV7-4 5′ GAA TTT ATG GAG TTT GGT CTG 3′ (SEQ ID NO:6)

Mutational Primers

HHV7-MUT55′ CAA AGC TGC AAC CCG GGG TTG TAG AAC A 3′ (SEQ ID NO:7)

HHV7-MUT6 5′ TGT TCT ACA ACC CCG GGT TGC AGC TTT G 3′ (SEQ ID NO:8)

Amplifled Sequences

SEQ ID NO:9. The section of SEQ ID NO:1 amplified by primers of SEQ IDNO:3 and SEQ ID NO:4

TTTTTA CATTTGGCTT GCTTTTTGGT TTGTAAATTC AATTGGACGG TTTGCTTAGA TTGCTGTGAAGCAAAGCTGC AAGACGGAGT TGTAGAACAT GCAACATTAA AGCTGTTGTA CTCTTCAGGCATGTTAGGAT GCAGACCAAA CTCCATAAAT TCTTTGGGAA GATAGGTACA GAAATAT

SEQ ID NO:10. The section of SEQ ID NO:1 amplified by primers of SEQ IDNO:5 and SEQ ID NO:6

T GCTTTTTGGT TTGTAAATTC AATTGGACGG TTTGCTTAGA TTGCTGTGAA GCAAAGCTGCAAGACGGAGT TGTAGAACAT GCAACATTAA AGCTGTTGTA CTCTTCAGGC ATGTTAGGATGCAGACCAAA CTCCATAAAT TC

SEQ ID NO:11. The section of SEQ ID NO:2 amplified by primers of SEQ IDNO:5 and SEQ ID NO:6

CAA AGCTGCAACC CGGGGTTGTA GAACA

11 193 base pairs nucleic acid double linear DNA (genomic) not provided1 AAGCTTTTTA CATTTGGCTT GCTTTTTGGT TTGTAAATTC AATTGGACGG TTTGCTTAGA 60TTGCTGTGAA GCAAAGCTGC AAGACGGAGT TGTAGAACAT GCAACATTAA AGCTGTTGTA 120CTCTTCAGGC ATGTTAGGAT GCAGACCAAA CTCCATAAAT TCTTTGGGAA GATAGGTACA 180GAAATATAAG CTT 193 183 base pairs nucleic acid double linear othernucleic acid /desc = “amplified control sequence” not provided 2TTTTTACATT TGGCTTGCTT TTTGGTTTGT AAATTCAATT GGACGGTTTG CTTAGATTGC 60TGTGAAGCAA AGCTGCAACC CGGGGTTGTA GAACATGCAA CATTAAAGCT GTTGTACTCT 120TCAGGCATGT TAGGATGCAG ACCAAACTCC ATAAATTCTT TGGGAAGATA GGTACAGAAA 180TAT 183 24 base pairs nucleic acid single linear other nucleic acid/desc = “PCR primer” not provided 3 TTTTTACATT TGGCTTGCTT TTTG 24 24base pairs nucleic acid single linear other nucleic acid /desc = “PCRprimer” not provided 4 ATATTTCTGT ACCTATCTTC CCAA 24 21 base pairsnucleic acid single linear other nucleic acid /desc = “PCR primer” notprovided 5 TGCTTTTTGG TTTGTAAATT C 21 21 base pairs nucleic acid singlelinear other nucleic acid /desc = “PCR primer” not provided 6 GAATTTATGGAGTTTGGTCT G 21 28 base pairs nucleic acid single linear other nucleicacid /desc = “PCR primer” not provided 7 CAAAGCTGCA ACCCGGGGTT GTAGAACA28 28 base pairs nucleic acid single linear other nucleic acid /desc =“PCR primer” not provided 8 TGTTCTACAA CCCCGGGTTG CAGCTTTG 28 183 basepairs nucleic acid double linear other nucleic acid /desc = “amplifiedtarget sequence” not provided 9 TTTTTACATT TGGCTTGCTT TTTGGTTTGTAAATTCAATT GGACGGTTTG CTTAGATTGC 60 TGTGAAGCAA AGCTGCAAGA CGGAGTTGTAGAACATGCAA CATTAAAGCT GTTGTACTCT 120 TCAGGCATGT TAGGATGCAG ACCAAACTCCATAAATTCTT TGGGAAGATA GGTACAGAAA 180 TAT 183 143 base pairs nucleic aciddouble linear other nucleic acid /desc = “amplified target sequence” notprovided 10 TGCTTTTTGG TTTGTAAATT CAATTGGACG GTTTGCTTAG ATTGCTGTGAAGCAAAGCTG 60 CAAGACGGAG TTGTAGAACA TGCAACATTA AAGCTGTTGT ACTCTTCAGGCATGTTAGGA 120 TGCAGACCAA ACTCCATAAA TTC 143 28 base pairs nucleic aciddouble linear other nucleic acid /desc = “amplified target sequence” notprovided 11 CAAAGCTGCA ACCCGGGGTT GTAGAACA 28

What is claimed is:
 1. A method of determining the amount of a targetnucleic acid having the sequence shown in SEQ ID NO:1 in a sample whichmethod comprises: (i) mixing the sample with a predetermined amount ofcontrol nucleic acid; (ii) bringing the mixture formed in (i) intocontact with a pair of nucleic acid molecules each comprising: a primerportion consisting of a contiguous sequence of from 10 to 50 nucleotidesthat hybridizes to (a) the target nucleic acid molecule represented bySEQ ID NO:1, or (b) to the complement of the molecule of SEQ ID NO:1 ina polymerase chain reaction (PCR) buffer comprising 16 mM (NH₄)₂SO₄, 67mM Tris-HCl (pH 8.8 at 25° C.), 0.01% Tween-20, 2 mM MgSO₄ at 50° C.;and optionally a further portion comprising from 1 to 25 nucleotidesjoined to and immediately 5′ to the 5′ end of the primer portion whereinone nucleic acid molecule has a primer portion of type (a) and the othernucleic acid molecule has a primer portion of type (b); and wherein thetwo primers in combination amplify the target nucleic acid moleculerepresented by SEQ ID NO:1, or a section thereof, in a polymerase chainreaction; (iii) performing a nucleic acid amplification reaction, saidreaction requiring the presence of the primer portions defined in (ii)to amplify the target nucleic acid sequence or a section thereof and thecontrol nucleic acid or a section thereof; (iv) determining the relativequantities of the amplified control and target nucleic acids; and (v)calculating from the determination of (iv) the amount of target nucleicacid in the sample.
 2. A method according to claim 1 whereinamplification is performed by means of a polymerase chain reaction(PCR).
 3. A method according to claim 2 wherein the control nucleic acidcomprises at least one restriction site not present in the targetsequence and wherein the determination performed in step (iv) comprisesthe step of cleaving one or more such restriction sites.
 4. A methodaccording to claim 3 wherein the restriction site is a SmaI site.
 5. Amethod according to claim 4 wherein the control nucleic acid comprisesthe sequence shown in SEQ ID NO:2 or any section thereof containing arestriction site not present in the sequence of SEQ ID NO:1.
 6. Themethod according to claim 1 wherein, in step (i), the nucleic acidmolecule of type (a) is the nucleic acid molecule of SEQ ID NO:3 and thenucleic acid molecule of type (b) is the nucleic acid molecule of SEQ IDNO:4; or the nucleic acid molecule of type (a) is the nucleic acidmolecule of SEQ ID NO:5 and the nucleic acid molecule of type (b) is thenucleic acid molecule of SEQ ID NO:6.
 7. The method according to claim 1wherein the pair of nucleic acid molecules of step (ii) respectivelyhybridize to the target nucleic acid sequence of SEQ ID NO:1 and itscomplementary strand in such positions that the 5′ end of each primerportion hybridizes at a location that is from 0 to 50 nucleotides 3′ tothe 5′ end of the sequence of SEQ ID NO:1 or the 5′ end of thecomplement of the nucleic acid molecule of SEQ ID NO:1.
 8. A methodaccording to claim 1 wherein contacting step (ii) and amplification step(iii) are performed at least one further time, each time using a furtherdifferent pair of nucleic acid molecules; the nucleic acid molecules ofthe pair used in each successive contacting and amplification routinehybridizing to the target sequence of SEQ ID NO:1 in such a positionthat the 5′ ends of the primer regions are located at least onenucleotide 3′ to the site at which the 5′ end of the previous pairhybridizes; such that the PCR performed is a nested PCR.
 9. A methodaccording to claim 8 wherein contacting step (ii) and amplification step(iii) are performed twice such that there is a first round of contacting(ii) and amplification (iii) and a second round, with the nucleic acidpair of claim 5 being employed in the first round and the nucleic acidpair of claim 6 being employed in the second round.
 10. The methodaccording to claim 8 wherein the first nucleic acid molecule of type (a)is the nucleic acid molecule of SEQ ID NO:3 and the first nucleic acidmolecule of type (b) is the nucleic acid molecule of SEQ ID NO:4. 11.The method according to claim 8 wherein the second nucleic acid moleculeof type (a) is the nucleic acid molecule of SEQ ID NO:5 and the secondnucleic acid molecule of type (b) is the nucleic acid molecule of SEQ IDNO:6.
 12. The method according to claim 8 wherein the different pair ofmolecules hybridizes to the target nucleic acid sequence of SEQ ID NO:1and its complementary strand in such positions that the 5′ end of eachprimer portion hybrizes at a location that is from 0 to 50 nucleotides3′ to the 5′ end of the sequence of SEQ ID NO:1 or the 5′ end of thecomplement of the nucleic acid molecule of SEQ ID NO:1.
 13. A kit forquantification of human herpesvirus 7 (HHV-7) in a sample, which kitcomprises: (i) a pair of nucleic acid molecules according to claim 6that amplifies both the target sequence and a control nucleic acidsequence, or sections thereof; and (ii) the control nucleic acidsequence.
 14. A kit according to claim 13 which further comprises (iii)a further pair of nucleic acid molecules according to claim 2 thathybridize to the target sequence of SEQ ID NO:1 in such a position thatthe 5′ ends of the primer portions of said further pair of nucleic acidmolecules are located at least one nucleotide 3′ to the site at whichthe 5′ end of said pair of primers according to claim 13 hybridizes. 15.A kit according to claim 14 comprising: (i) a control sequence of SEQ IDNO:2, (ii) a pair of nucleic acids of SEQ ID NO:3 and SEQ ID NO:4, and(iii) a pair of nucleic acids of SEQ ID NO:5 and SEQ ID NO:6.
 16. Thekit according to claim 13 wherein the control nucleic acid comprises atleast one restriction site not present in the target sequence.
 17. Thekit according to claim 16 wherein the restriction site is a SmaI site.18. The kit according to claim 17 wherein the control sequence comprisesthe sequence shown in SEQ ID NO:2 or any section thereof containing arestriction site not present in the sequence of SEQ ID NO:1.
 19. The kitaccording to claim 13 wherein the pair of nucleic acid moleculeshybridizes to the target nucleic acid sequence of SEQ ID NO:1 and itscomplementary strand in such positions that the 5′ end of the eachprimer portion hybridizes at a location that is from 0 to 50 nucleotides3′ to the 5′ end of the sequence of SEQ ID NO:1 or the 5′ end of thecomplementary strand thereto.
 20. The kit according to claim 13 whereinthe nucleic acid molecule of type (a) is the nucleic acid molecule ofSEQ ID NO:3 and the nucleic acid molecule of type (b) is the nucleicacid molecule of SEQ ID NO:4.
 21. The kit according to claim 13 whereinthe nucleic acid molecule of type (a) is the nucleic acid molecule ofSEQ ID NO:5 and the nucleic acid molecule of type (b) is the nucleicacid molecule of SEQ ID NO:6.
 22. A pair of nucleic acid molecules eachcomprising: (i) a primer portion consisting of a contiguous sequence of10 to 50 nucleotides which hybridizes to (a) a target nucleic acidmolecule represented by SEQ ID NO:1, or (b) the complement of thenucleic acid molecule of SEQ ID NO:1 in a polymerase chain reaction(PCR) buffer comprising 16 mM (NH₄)₂SO₄, 67 mM Tris-HCl (pH 8.8 at 25°C.), 0.01% Tween-20, 2 mM MgSO₄ at 50° C.; and optionally (ii) a furtherportion comprising from 1 to 25 nucleotides joined to and immediately 5′ to the 5′ end of the primer portion; wherein one nucleic acid moleculecomprises a primer portion of type (a) and the other nucleic acidmolecule comprises a primer portion of type (b); and wherein the twoprimers in combination amplify the target nucleic acid moleculerepresented by SEQ ID NO:1, or a portion thereof, in a polymerase chainreaction.
 23. A pair of nucleic acid molecules according to claim 22wherein the nucleic acid molecule of type (a) is the nucleic acidmolecule of SEQ ID NO:3 and the nucleic acid molecule of type (b) is thenucleic acid molecule of SEQ. ID NO:4.
 24. A pair of nucleic acidmolecules according to claim 22 wherein the nucleic acid molecule oftype (a) is the nucleic acid molecule of SEQ ID NO:5 and the nucleicacid molecule of type (b) is the nucleic acid molecule of SEQ. ID NO:6.25. A pair of nucleic acid molecules according to claim 2 that,respectively, hybridize to the target nucleic acid sequence of SEQ IDNO:1 and its complementary strand in such positions that the 5′ end ofthe each primer portion hybridizes at a location that is from 0 to 50nucleotides 3′ to the 5′ end of the sequence of SEQ ID NO:1 or the 5′end of the complement of the nucleic acid molecule of SEQ ID NO:1.
 26. Anucleic acid molecule selected from the group consisting of 5′ TTT TTACAT TTG GCT TGC TTT TTG 3′ (SEQ ID NO:3); 5′ ATA TTT CTG TAC CTA TCT TCCCAA 3′ (SEQ ID NO:4); 5′ TGC TTT TTG GTT TGT AAA TTC 3′ (SEQ ID NO:5);and 5′ GAA TTT ATG GAG TTT GGT CTG 3′ (SEQ ID NO:6).
 27. An isolatednucleic acid molecule selected from the group consisting of thesequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:10and SEQ ID NO:11 (CAAAGCTGCAACCCGGGGTTGTAGAACA); and the complementarystrands thereof.